Method promoting conception by administering IL-8 or MCAF

ABSTRACT

Leukocyte chemotactic factors such as interleukin-8 and MCAF, and inductive substances therefor were revealed to have proconceptive activities. These activities include promoting ovum growth and fertilized ovum implantation, and are exhibited by a sole substance. Accordingly, it has been shown that such substances can be used in drugs concerning medical treatment for infertility, and further, can be used in veterinary and livestock industry fields such as reproduction of industrial animals or species preservation of rare animals.

TECHNICAL FIELD

The present invention relates to a proconceptive agent including aleukocyte chemotactic factor or a substance inducing the leukocytechemotactic factor as an active principle which has the function ofpromoting growth of ova and ovarian follicles together with the functionof promoting implantation of a fertilized ovum by promoting thickeningof endometrium and softening of the interstitial tissue due toedematization. In particular, the present invention relates to aproconceptive medical treatment for mammals.

BACKGROUND ART

In general, the following conditions are considered as essential forestablishment of pregnancy:

(1) The existence of sufficient number of sperms having sufficientmotility and fertility in an ejaculated seminal fluid;

(2) Normal growth and maturation of ovarian follicles and ova, anddischarging of an ovum in ovulation which has fertilizability andcleaving ability;

(3) The form and function of an oviduct maintained normally; and

(4) The form and function of a uterus being normal for embryoimplantation and maintenance thereof.

In the present situation, however, at least one out of ten marriedcouples is estimated as being unable to satisfy one or moreabove-described conditions, namely, is infertile. The cases can bebroadly classified into 40% of the cases where the causes can beattributed to female factors; 25% of the cases where the causes can beattributed to male factors; 25% of the cases where the causes can beattributed to both female and male factors; and 10% of cases where thecauses cannot be clarified.

In respect of such causes, incurable cases as described below are called"absolute infertility": No ova to be discharged are present; the uterushas been extirpated; the husband is azoospermia; or the like. Hitherto,although the case in which both oviducts had been extirpated wasregarded as absolute infertility, pregnancy and birth even in such caseshave become possible by means of in vitro fertilization (IVF) or embryotransfer (ET). According to development of such an in vitrofertilization or embryo transfer method, pregnancy in cases such asatretic oviduct or oligozoospermia, in which pregnancy had beenimpossible, has become possible. The ratio of pregnant cases by in vitrofertilization to the total number of in vitro fertilization operationsis still as low as approximately 20 to 30%, and the ratio of the casesresulting in birth is only 5 to 10%. Concerning such low ratios,implantation failure in returning fertilized ova to the uterus isconsidered as a cause. In addition, implantation disability is pointedout as possibly occupying a high percentage of the causes forunaccountable infertility or so called functional infertility.

Although artificial insemination is also frequently performed in animalsother than human beings, the conception rate and birth rate therein arenot necessarily satisfactory. In particular, when such operations areperformed for preservation and reproduction of rare animals, such lowrates are problems since there exists a temporal limitation due to thehigh ages of the remaining animals or the like. Similar to cases ofhuman beings, it is considered that such low rates may be attributed toa failure in sufficiently satisfying the above-described conditions inthe steps for establishing pregnancy.

Through a series of developmental stages, an ovum is formed from anoocyte which has been derived from an oogonium. In mammals, an oogoniumstops dividing just before or just after birth, and transforms into anoocyte which is a meiotic-type cell. In the latter stage of the prophaseof the first meiosis, the oocyte comes into a stationary phase, andchanges in its chromosomes do not progress (interphase of the firstmeiosis). At this time, the nucleic volume of the oocyte increases, andsuch an oocyte is called a "protoblast" (or "germinal vesicle"). Theoocyte, then, restarts meiosis in response to gonadotropin secreted fromthe pituitary gland. In many mammals, the meiosis is restarted in anovarian follicle before ovulation; the protoblast is then broken(germinal vesicle breakdown); a first polar body is released through themetaphase, the anaphase and telophase of the first meiosis; and thechromosomes stop changing in the metaphase of the second meiosis(interphase of the second meiosis). According to fertilization, thesecond meiosis restarts, a second polar body is released, and themeiosis is thus completed ["Jikken Seishoku-Seirigaku no Tenkai(Development of Experimental Reproductive Physiology)", vols. 11 to 16edited by Yosisuke Suzuki, and published by Soft Science Co., Ltd,1982]. Investigations on ovum maturation have been conducted principallyusing in vitro culture systems while employing the following threemethods depending on the purposes of experiments.

(1) Culturing oocytes harvested from ovarian follicles under severalconditions. This method is employed for analysis of factors influentialupon ovum maturation.

(2) Culturing oocytes together with theca cells, or culturing oocytes ina conditioned medium based on ovarian follicles. This method is employedfor analyzing influences by theca cells.

(3) So called "organ culture" in which ovarian follicles includingoocytes are externally cultured. This method is employed for analysis offactors influential upon ovum maturation passing through or withintervention of thecae.

As the first step for conception, an adequate maturation process for avital ovum is essential. The term "ootid" means an ovum which canrelease a second polar body in response to fertilization, and therebycomplete fertilization as well as meiosis (namely, havingfertilizability), and which can develop in response to fertilizationstimulation (namely, having developmental potency).

The latter step for conception includes the activity of a fertilizedovum traveling through an oviduct toward the inner surface of a uterusfor implantation. When a disability concerning either of theabove-described steps is present on the female side, conception cannotbe achieved or can be achieved only with a markedly low degree ofprobability though depending on the degree of the disability.Implantation is understood as an adhesion phenomenon between afertilized ovum and endometrium. Upon implantation, endometrium as areceiver for a fertilized ovum should sufficiently thicken, and theinterstitial tissue should be edematized to be soft. It was reportedthat implantation of a fertilized ovum rarely occurs if the interstitialtissue does not swell in the ovulation period. In the above-describedinfertility cases in human beings, several therapeutic treatments areperformed depending on the causes thereof. For example, foroligozoospermia or azoospermia, sperms manually obtained are injected onan ovulation day into the uterus lumen or the oviduct in an attempt toachieve natural fertilization in the oviduct. Nowadays, a justejaculated seminal fluid is not used for fear of bacterial infection,and therefore, washed and condensed sperms are used. In many cases,however, a few to tens of attempts at insemination are required untilconception is achieved, and unsuccessful implantation is considered asone reason for such requirement.

Further, in vitro fertilization and embryo transfer are performed incases such as atretic oviduct or oligozoospermia where othertherapeutical treatments are not effective, wherein ova taken out of abody are inseminated with sperms, and after fertilization, divided andgrown embryos are transvaginally transferred on the inner surface of auterus. In practice, the operation comprises the following significantsteps: (1) Induction of superovulation, and determination of a ovumcollection period; (2) an operation for ovum collection; (3)supplemental culturing for ovum maturation; (4) collection ofsatisfactory sperms and capacitation; (5) in vitro fertilization; (6)culturing of the fertilized ova; (7) embryo transfer; and (8) lutealphase management. Without completion of these steps, pregnancy cannot beexpected to occur. Since the endocrine system is not physiologicallynatural in such an estrus cycle with superovulation induction,hormonotherapy as an luteal phase management, such as administration ofprogesterone or the like for approximately one week after the embryotransfer, is performed for luteinization, namely thickening ofendometrium and softening of the interstitial tissue. Nevertheless, thepregnancy rate is unsatisfactory, as described above, and therefore,there are demands for a further improved method.

As a modification of in vitro fertilization/embryo transfer, a relatedart generically called "assisted reproductive technology" has beendeveloped. This technology includes intraoviduct zygote transplantationin which in vitro fertilization is performed, and then fertilized ova,either before division or after division and growth, are transplantedinto an oviduct; and intraoviduct gametes transplantation in whichcollected ova in an early phase are transplanted together with spermsinto an oviduct. Although these are methods which provide conditionscloser to the physiologically natural process of pregnant, problemsconcerning implantation of a fertilized ovum has not yet been solved,and there is room for improvement.

Meanwhile, inflammation reactions as bioprotective reactions areobserved not only in bacterial or viral infections and injuries, butalso in tissue damage due to autoimmune reactions. At this time,specific peripheral leukocytes infiltrate into the inflammatory region.For leukocyte migration on such occasions, chemokines (chemotacticfactors) play an important role. Interleukin-8 is one of suchchemokines, and its excessive production is considered as a cause ofseveral inflammatory diseases.

Interleukin-8 has been reported to be produced from several types ofcells such as fibroblasts and several tumor cells as well as hemocytessuch as monocytes, macrophages, and lymphocytes in response tostimulation by IL-1, TNF, LPS, or the like. Accordingly, interleukin-8is deduced to be an important mediator for acute inflammation, andabnormal production is considered relating to some diseases. Examples ofsuch diseases include rheumatoid arthritis, gouty arthritis, asthma,septicemia, immunological vasculitis, hepatitis, and pyelonephritis. Theactivity of interleukin-8 in such diseases has, however, been known onlyat a basic science, and the mechanism of how it relates to progress inpathologic processes of such diseases has not yet been clarified.Interleukin-8 is a chemokine which has the functions of causingchemotaxis of neutrophils and lymphocytes, and activating neutrophilsand other mononuclear leukocyte or the like. The term "chemokine" meansa bioactive substance (or cytokine) which has an activity of causingchemotaxis. Interleukin-8 is a protein comprising 69 to 77 amino acids.The number of amino acids alters depending on the situation since the Nterminus processed with intra- or extracellular enzymes. At first,interleukin-8 was reported in the name of MDNCF (K. Matsushima, et al.,J. Exp. Med., 167, 1883, 1988), after that, it was also found by otherresearchers and named differently, and the name was unified as"interleukin-8" (C. G. Larsen, et al., Science, 243, 1464, 1989).Interleukin-8 belongs to the CXC chemokine family, the members of whichhave similar amino acid numbers and similar cysteine residueconfigurations. "CXC chemokine family" is a generic term for a group oflow-molecular weight proteins which mutually have amino acid homologiesof approximately 30%, and the same four cysteine residue positions.Structural analogues of interleukin-8 such as γIP-10, GRO (α, β, γ),PF-4, and NAP-10 are known as proteins belonging to this family otherthan interleukin-8 (N. Mukaida, et al., Microbiol. Immunol., 36, 773,1992). Proteins of the CXC chemokine family are characterized by havinga N terminus amino acid sequence in which two cysteine residues arebonded with an intervening amino acid residue. "CXC chemokine" is alsocalled "β chemokine" (J. J. Oppenheim, et al., Annual. Rev. Immunol., 9,617, 1991).

Monocyte chemotactic and activating factors (hereinafter referred to asMCAF) have been reported to be produced from several types of cells suchas fibroblasts, endothelial cells, smooth muscle cells and several tumorcells as well as hemocytes such as monocytes, macrophages, andlymphocytes in response to stimulation by IL-1, TNF, IFN-γ, LPS, or thelike. Additionally, the monocyte chemotactic and activating factor isalso called MCP-1 (monocyte chemoattractant protein-1) or GDCF(glioma-derived monocyte chemotactic factor), and is a proteincomprising 76 amino acids and having 4 cysteine residues. Reports havebeen made concerning identification and gene cloning of MCAF, MCP-1 orGDCF (K. Matsushima, et al., J. Exp. Med., 169, 1485, 1989; Y. Furutani,et al., Biochem. Biophys. Res. Commun., 159, 249, 1989; E. R. Robinson,et al., Proc. Natl. Acad. Sci. USA, 86, 1850, 1989; and T. Yoshimura, etal., FEBS Letters, 244, 487, 1989). Hereinafter, in the presentinvention, "MCAF" is used as a generic term also including MCP-1 andGDCF.

MCAF belongs to the CC chemokine family, the members of which havesimilar amino acid numbers and similar cysteine residue configurations."CC chemokine family" is a generic term for a group of low-molecularweight proteins which mutually have amino acid homologies ofapproximately 30%, and the same four cysteine residue positions.Structural analogues of MCAF such as RANTES, LD78, ACT2, I-309, MCP-2and MCP-3 in human beings, or JE, MIP-1 α, MIP-1 β, and TCA-3 in miceare known as proteins belonging to this family other than MCAF (N.Mukaida, et al., Microbiol. Immunol., 36, 773-789, 1992). Proteins ofthe CC chemokine family are characterized by having a N terminus aminoacid sequence in which two cysteine residues are bonded in series. "CCchemokine" is also called "β chemokine" (J. J. Oppenheim, et al.,Annual. Rev. Immunol., 9, 617, 1991).

The present inventors have reported that administration of aninterleukin-8 suppository to a rabbit can cause cervical ripening (ElMaradny, et al., Am. J. Obstet. Gynecol., 171, 77, 1994). Cervicalripening is essential for parturition, and cervical ripeningincompetency is the principal cause of today's dystocia. By localadministration of interleukin-8, inflammatory cells such as neutrophilsmigrate into the cervix to release collagenase, elastase or the likewhich degrades collagen in the interstitial tissue, increase the contentof water in the interstitial tissue, and thus cervical ripening takesplace. However, effects of interleukin-8 or MCAF upon the steps ofoogenesis, ovum maturation, fertilization, and achievement of conceptionhave not yet been known.

The following are known as examples of means for improving conceptionrate: Inductive substances for resumption of the first meiosis such ascyclic AMP, calcium ions, prostaglandins, cholera toxins, and forskolin;follicle stimulating hormone (FSH) or the like for attempting follicularmaturation; estrogen or the like for attempting to provide developmentalpotency for fertilized ova; and luteal phase management using a steroidagent or the like for attempting to achieve implantation of fertilizedova.

According to these means, however, a satisfactory pregnancy rate may notbe achieved, or some agents cannot actually be used or can be used withlimitations due to side effects inherent in the uses thereof.

DISCLOSURE OF INVENTION

The present invention relates to a proconceptive agent including aleukocyte chemotactic factor or an inducer thereof as an activeprinciple.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of Example 1. Interleukin-8 is administrated togroup A while saline is administrated to group B. The horizontal axisindicates the number of fetuses per maternal body while the verticalaxis indicates the number of maternal bodies which had fetuses of thenumber indicated by the horizontal axis.

FIG. 2 indicates the results of Example 5.

FIG. 2(A) shows a hematoxylin/eosin-stained section of an ovary as acontrol (at 0 hours and without drug administration);

FIG. 2(B) shows a hematoxylin/eosin-stained section of an ovary 10 hoursafter the administration of hCG; and

FIG. 2(C) shows a hematoxylin/eosin-stained section of an ovary 10 hoursafter administration of interleukin-8. The bars in these picturesindicate 200 μm.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors gave attention to the facts that cytokines areinvolved in the ovulation process for pregnancy, and that theendometrium is in a physiological inflammatory state during the processof fertilized ovum implantation. As a result, we found thatinterleukin-8 or MCAF, which cause migration and activation ofleukocytes, exhibit suitable effects in each step for achievingpregnancy (growth of ovarian follicles, resumption of meiosis,implantation of a fertilized ovum, and others), and administrationsolely of such an agent can improve conception rate. Based on thesefindings, the Inventors accomplished the present invention. In thepresent invention, the proconceptive action includes both steps forpromoting maturation of unfertilized ova, and promoting implantation offertilized ova.

Promotion of fertilized-ovum implantation, which is the latter step ofthe present invention, comprises local administration of a leukocytechemotactic factor to cause a biological reaction aiming at localmigration of leukocytes. The term "leukocyte chemotactic factor" is ageneric term for all substances capable of inducing leukocytechemotaxis, and includes several substances produced in somemicroorganisms or inflammatory tissues. Examples of such substancesinclude interleukin-8 and chemokines analogous thereto. The chemokinesanalogous to interleukin-8 include chemokines belonging to theinterleukin-8 family, namely, CXC chemokines (such as GCP-2, GROα, GROβ,GROγ, PF4, NAP-2, ENA-78, IP-10, MIG, PBP, CTAP-III, SDF-1α, andSDF-1β); and chemokines belonging to the MCAF (or MCP-1, i.e. monocytechemotactic and activating factor or monocyte chemoattractant protein-1)family, namely, CC chemokines (such as hMIP-1α, hMIP-1β, MCP-2, MCP-3,RANTES, and I-309) (Drug & Development, 7, 57-65, 1996). MCAF is knownto cause physiological inflammation by causing migration and activationof monocytes and macrophages, and secondary production of interleukin-8during this process has been observed. Accordingly, MCAF and analoguesthereof having such physiological activities can also be used forpromoting fertilized-ovum implantation since they can indirectly thickenthe endometrium and soften the interstitial tissues. From among suchsubstances, interleukin-8 and MCAF are preferably used.

Additionally, the following substance may also be preferably used:Substances inducing production of a leukocyte chemotactic factor, suchas interleukin-1, interferon γ, tumor necrosis factors (TNF); plateletactivating factors (PAF) having physiological activities analogous tochemokines and serving as leukotactic substances; and LPS, which is oneendotoxin.

Interleukin-8 and MCAF can be obtained from natural sources according toordinary isolation/purification methods such as column chromatography.Alternatively, those obtained by culturing cells which produce them,chemical synthesis, or gene recombination technology can also be used.Such production methods are disclosed in Japanese Laid-open PatentPublications(Kohyo) No. 3-505037 and No. 4-500156, Japanese UnexaminedPatent Publication(Kokai) No. 2-207788, and others. Additionally,commercially available interleukin-8 and MCAF manufactured by companiessuch as Oncogene Science (USA) and PEPROTEC (USA) can also be used.

Further, the origin of the interleukin-8 or MCAF to be used is notlimited to human beings or animals which are the subjects to beadministered. Interleukin-8 or MCAF species derived from dissimilaranimal origin can be used so long as the problems concerning undesiredeffects upon the human or animal body to be administered, such ashyperimmune response, can be overcome. Moreover, those having the sameamino acid sequences except for one or more amino acid residues whichare replaced, inserted, deleted, and/or modified; and peptides eachcomprising a part of the amino acid sequence for the objective structurecan also be used for working of the present invention so long as theyexhibit the desired activity.

Incidentally, interleukin-8 or MCAF can be solely administrated directlyinto the uterus, and in addition, combined formulations comprising boththereof or ingredients having identical or analogous effects can also beadministrated.

Additionally, ordinarily used excipients, binders, lubricants,tinctions, stabilizers, and others can be added to the medicine of thepresent invention in the preparation process. The dosage form of theproconceptive agent in relation to the present invention is notespecially limited so long as the object of the present invention can beachieved thereby, and thus, the dosage form can be a liquid type such asa liquid formulation to be sprayed in the uterus; a semi-solid type suchas an ointment, a cream, or a gel formulation; and a solid type such asa virginal tablet, a vaginal capsule, a pessary, or a vaginalsuppository. Further, when a formulation such as a liquid or semi-solidformulation which can be applied onto a uterine cervix dilator isemployed, administration through such a dilator can be performed.Hereupon, the action "application" includes not only application of themedicine onto the surface of such a dilator or the like, but alsoimmersion of the medicine onto such a dilator or the like through aimmersing means properly provided thereon. In addition, intraperitonealadministration through the Douglas pouch or the like can be carried out.As a matter of course, dosage forms suitable to intraperitoneal,intravenous, oral, or transpulmonary administration other than localadministration can be employed for achieving the purpose of the presentinvention, as typically shown in examples of the present inventiondescribed below.

In preparation of the above-described formulations, additive ingredientsgenerally used in drug formulations can be combined to achieve theobjective dosage forms.

Examples of such ingredients include animal or vegetable oils (such assoybean oil, coconut oil, beef tallow, and synthetic glyceride);hydrocarbons (such as liquid paraffin, squalene, and solid paraffin);ester oils (such as octyl dodecyl myristate and isopropyl myristate);higher alcohols (such as cetosterial alcohol and behenil alcohol);silicon resins; silicon oils; surfactants (such as polyoxyethylene fattyacid esters, sorbitan fatty acid esters, glycerol fatty acid ester,polyoxyethylene fatty acid esters, polyoxyethylene hardened castor oil,and polyoxyethylene polyoxypropylene block copolymer); water-solublepolymers (such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinylpolymer, polyethylene glycol, polyvinyl pyrrolidone, and methylcellulose); alcohols (such as ethyl alcohol, and isopropyl alcohol);polyhydric alcohols (such as glycerol, propylene glycol, dipropyleneglycol, and sorbitol; saccharides (such as glucose and sucrose);inorganic powders (such as silicic acid anhydride, aluminum magnesiumsilicate, and aluminum silicate); and purified water. For pH adjustment,compounds such as described below can be used: Inorganic acids (such ashydrochloric acid and phosphoric acid); alkali metal salts of inorganicacids (such as sodium phosphate); inorganic bases (such as sodiumhydroxide); organic acids (such as lower fatty acids, citric acid, andlactic acid); alkali metal salts of organic acids (such as sodiumcitrate and sodium lactate); and organic bases (such as arginine andethanolamine). As occasion demands, preservatives, antioxidants, andothers can also be added. In practice, such additives are selected,optionally or in combination, from among the above-described compoundsdepending on the dosage form of the medicine for the object disease ofthe present invention, though, needless to say, not limited to theabove-described compounds. Additionally, formulations satisfying theobject of the present invention can readily be obtained by mixing theactive principle in a commercially available suppository basis (such asWitepsol manufactured by Mitsuba Co., Ltd.). Further, the material andshape of the uterine cervix dilator to be used in the present inventionis not especially limited so long as the dilator can be used for thedesired object. Examples of such dilators include dilators comprising aplant such as Laminaria japonica; dilators comprising conventionallyused synthetic resins (water-absorptivity, thermoplasticity, andwater-bloating tendency of the resin do not matter, and whether the basematerial is spongiform or fibrous does not matter either); and dilatorscomprising polymeric absorbents.

Interleukin-8 and MCAF are administrated at, for example, 1 pg/kg to 10mg/kg, preferably 100 pg/kg to 1 mg/kg, and more preferably 1 ng/kg to10 μg/kg though it depends on the age of the woman as an administrationsubject, the state of reproductive system such as ovary function anddegree of thickening in the endometrium, and health conditions of thematernal body. The above-described ranges are not directed to anylimitation, and interleukin-8 and MCAF may be contained in amountsnecessary to exhibit the desired pharmacological effects. Further, thedosages of other leukocyte migration factors or inducers therefor shouldfinally be determined by the medical staff.

The present invention provides a usage of the compound of the presentinvention in relation to pregnancy. Further, the present inventionprovides a usage of the compound of the present invention in relation tointernal, surgical, or diagnostic treatments for ovum maturation andpromotion of fertilized ovum implantation. Accordingly, the presentinvention can be utilized in human beings as described below, though notlimited to such uses.

(1) In infertility cases with no abnormalities both in sperm and ova,such as functional infertility, the compound of the present inventioncan be administrated before or after copulation in an attempt to promotethe growth of ova and/or to promote fertilized ovum implantation,thereby enhancing the possibility of pregnancy or conception.

(2) In infertility cases where the causes are attributed to either orboth male and/or female factors, or infertility cases where the causesare unclarified, the compound of the present invention can be used foran in vitro ovum treatment, and/or administrated to the maternal body atthe vagina, uterus and neighboring areas thereof before and after, or atthe same time as such a treatment in artificial insemination or humanbeing in vitro fertilization in an in vitro fertilization/embryotransfer method or a modification method thereof such as intraoviductzygote transplantation and intraoviduct gametes transplantation, in anattempt to promote growth of ova and/or to promote fertilized ovumimplantation, thereby enhancing the possibility of pregnancy orconception.

The invention described above is applicable to animals other than humanbeings. Specifically, a proconceptive agent containing interleukin-8 orMCAF as an active principle is used for in vitro treatment of ovacollected beforehand, and/or directly administrated to the animal forovum growth, or as a therapy, operation or treatment concerning afertilized ovum implantation process. In other words, such an agent canbe used for the purpose of reproduction of domestic animals such ascattle, horses, and pigs; companion animals such as dogs and cats; andwild animals such as giant pandas, tigers, and rhinoceroses.Reproduction of these animals is beneficial to mankind since it leads tolow-cost provision of meat and milk from superior domestic animals,maintenance of stocks exhibiting excellent characteristics, andpreservation of rare animals. For animal use, the compound of thepresent invention can be used as described below, though not limited tosuch uses.

(1) The compound of the present invention is administrated before orafter copulation of animals in an attempt to promote the growth of ovaand/or to promote fertilized ovum implantation, thereby enhancing thepregnancy or conception rate.

(2) The compound of the present invention is used for an in vitrotreatment of ova collected from the maternal body, and/or administratedto the maternal body at the vagina, uterus and neighboring areas thereofon artificial insemination or in vitro fertilization of an animal in anattempt to promote growth of ova and/or to promote fertilized ovumimplantation, thereby promoting achievement of pregnancy or conception.

EXAMPLES

The effects of the present invention are shown with reference toexamples below. The following examples are described only forillustration, and should not be understood as limitations in anyrespect. Incidentally, the interleukin-8 and MCAF provided by Dr. K.Matsushima were used in the following examples (K. Matsushima, and J. J.Oppenheim, CYTOKINE, 1, 2-13, 1989).

Example 1 Promoting Effect of Interleukin-8 on Fertilized OvumImplantation

The following experiment using rats was conducted in order to evaluatethe effect of interleukin-8 on fertilized ovum implantation.

(1) Method

Fourteen Wister rats (clean grade, female, 10-week-old) were dividedinto two groups, A and B, each comprising 7 rats. The female rats ofgroup A (interleukin-8 dosed group) were placed in cages one by one, andmale Wister rats (clean grade) were then placed in the same cages one byone. Every morning, whether or not mating had occurred was confirmedaccording to vaginal smear observation, and the rat couples wereseparated when completion of mating was confirmed. Immediately aftermating confirmation, the female rats were intraperitoneally dosed with10 μg/0.2 ml saline of interleukin-8 each, and observed for 10 days. Onthe tenth day, the female rats were dissected, and the number of fetuseswere determined. Similarly, the female rats of the group B (controlgroup) were placed in cages one by one, and male Wister rats (cleangrade) were then placed in the same cages one by one. Every morning,whether mating had occurred or not was confirmed according to vaginalsmear observation, and the rat couples were separated when completion ofmating was confirmed. Immediately after mating confirmation, the femalerats were intraperitoneally dosed with 0.2 ml of saline each, andobserved for 10 days. On the tenth day, the female rats were dissected,and the number of fetuses were determined.

(2) Results

The rat uterus is bicornuate, and in general, the number of fetuses perfemale rat is approximately 9 to 11. The number of fetuses in group Awas 12±0.6 while that in group B was 10±1.4, and accordingly, anincrease in the number of fetuses was observed in the group dosed withinterleukin-8 (P<0.068). The histograms of groups A and B are shown inFIG. 1. In the group dosed with interleukin-8, every maternal rat exceptfor one rat got pregnant with 12 or more fetuses, and accordingly, animprovement in conception rate was clearly recognized. Incidentally,miscarriage due to administration of interleukin-8 was not observed inany rats.

Example 2 Observation of Histological Changes in Rats Dosed withInterleukin-8

The following experiment using rats was conducted in order tohistologically observe the effects of interleukin-8 on the endometria.

(1) Method

Six Wister rats (clean grade, female, 10-week-old) were divided into twogroups, A and B, each comprising 3 rats. Each rat in group A(interleukin-8 dosed group) was intraperitoneally dosed with 10 μg/0.2ml saline of interleukin-8 each, and 12 hours later, the uterus and theendometrium were visually inspected. These tissues were then fixed witha 10% neutral buffered formalin and stained with hematoxylin/eosin, andhistological changes were microscopically observed. Similarly, each ratin group B (control group) was intraperitoneally dosed with 0.2 ml ofsaline alone, and 12 hours later, the uterus and the endometrium werevisually inspected. These tissues were then fixed with a 10% neutralbuffered formalin and stained with hematoxylin/eosin, and histologicalchanges were microscopically observed.

(2) Results

The endometrium of each rat in group A was markedly swollen to beedematous, and thickening was also recognized. This state of theendometrium is similar to that of a human being in case of fertilizedovum implantation. On the other hand, changes such as swelling orthickening of the endometrium could not be observed in group B (controlgroup).

Example 3 Observation of Histological Changes in the Endometria ofRabbits Dosed with Interleukin-8

The following experiment using rabbits was conducted in order tohistologically observe the effects of interleukin-8 on the endometria.

(1) Method

Six New Zealand white rabbits (female, body weight of 3 kg) were dividedinto two groups, A and B, each comprising 3 rabbits. Each rabbit ofgroup A (interleukin-8 dosed group) was intravaginally dosed with asuppository every day for 3 days, wherein the suppository had beenprepared by adding 1 μg of interleukin-8 to 100 μl of 50° C.-meltedWitepsol W35 (Mitsuba Co., Ltd.), and by cool-solidifying the resultantat 4° C. On the fourth day from the start of the experiment, dissectionwas performed, and the isolated uteri were visually inspected and thenfixed with a 10% neutral buffered formalin. The obtained tissue sectionswere stained with hematoxylin/eosin, and histological changes weremicroscopically observed. Similarly, each rabbit in group B (controlgroup) was intravaginally dosed with a suppository which comprised thebase alone and was prepared in the same manner as for the group A, andsubjected to dissection and observation of histological changes in thesame manner as for group A.

(2) Results

Similar to the results of Example 2, the endometrium of each rabbit ingroup A was markedly swelled to be edematous, and thickening was alsorecognized. It was proven that administration of the drug in the mannerof a suppository could also induce endometrial changes similar to thoseshown in fertilized ovum implantation. On the other hand, such changescould not be observed at all in group B (control group).

Example 4 Observation of Histological Changes in Rats Dosed with MCAF

The following experiment was conducted in order to histologicallyobserve the effects of MCAF on the endometria of rats.

(1) Method

Six Wister rats (clean grade, female, 10-week-old) were divided into twogroups, A and B, each comprising 3 rats. Each rat in group A (MCAF dosedgroup) was intraperitoneally dosed with 10 μg/0.2 ml saline of MCAF, and12 hours later, the uterus and the endometrium were visually inspected.These tissues were then fixed with a 10% neutral buffered formalin andstained with hematoxylin/eosin, and histological changes weremicroscopically observed. Similarly, each rat in group B (control group)was intraperitoneally dosed with 0.2 ml of saline alone, and 12 hourslater, the uterus and the endometrium were visually inspected. Thesetissues were then fixed with a 10% neutral buffered formalin and stainedwith hematoxylin/eosin, and histological changes were microscopicallyobserved.

(2) Results

The endometrium of each rat in the group A was markedly swelled to beedematous, and thickening was also recognized. This state of theendometrium is similar to that of a human being in case of fertilizedovum implantation. On the other hand, changes such as swelling orthickening of the endometrium could not be observed in the group B(control group).

Example 5 Effect of Interleukin-8 (in vitro Administration) on Growth ofOvary Oocytes

(1) Method

This experiment was carried out according to a basic procedure asfollows: Twelve Wister rats (clean grade) which were sexually immature(27- to 28-day-old) were each dosed with 15 international units ofpregnant mare serum gonadotropin (PMSG, Sigma, St. Louis, Mo.), and 48hours later, intraperitoneally dosed with 15 international units ofhuman chorionic gonadotropin (hCG, Sigma, St. Louis, Mo.) or 17 μg ofinterleukin-8.

After the administration of hCG or interleukin-8, ovary extirpation wasperiodically performed, and the isolated ovaries were fixed with 3.5%paraformaldehyde (pH 7.4) for 24 hours. The resultant was thendehydrated with a series of increasing concentration alcohol solutions,embedded in paraffin, sliced into sections of 4 μm in thickness,deparaffinized, hydrated, and stained with hematoxylin/eosin. Thestained sections were subjected to microscopic examination forobservation of the nucleic stage in meiosis and cumulus-oocyte complexesin Graafian follicles. Resumption of meiosis was confirmed according tobreakdown of the protoblast (germinal vesicle) from each oocyte.

(2) Results

FIG. 2 contains section views of the maximum diameter parts of isolatedovaries. Before hCG administration, all oocytes in normal Graafianfollicles had normal germinal vesicles (FIG. 2A). Ten hours after hCGadministration, the germinal vesicles disappeared, and most of theoocytes had extruded first polar bodies. As is shown FIG. 2B and obviouswhen compared with the control, ovarian follicles of the rats hadmarkedly developed with hCG administration. These results are consistentwith results which have been reported previously. On the other hand,ovarian follicles also grew by administration of interleukin-8 insteadof hCG to a degree similar to that by hCG administration (FIG. 2C). WithhCG administration, 72% of germinal vesicles came into breakdown while54% of germinal vesicles came into breakdown with interleukin-8administration.

Additionally, the number of oocytes and the number of germinal vesiclebreakdowns after hCG or interleukin-8 administration are shown in Tables1 and 2.

                  TABLE 1                                                         ______________________________________                                        Germinal Vesicle Breakdown in Rat Graafian                                    Follicle Ova after PMSG and hCG Administration                                Time after hCG                                                                            Number of  Germinal Vesicle Breakdown                             Administration (hour)                                                                     Observed Ova                                                                             Number    Percentage                                   ______________________________________                                         0 [n = 1]  51         2            4                                         10   [n = 3]                                                                                    114           82                                                                                      71.9                                20 [n = 1]                     25                                                                                       52                                  ______________________________________                                         The examined graafian follicles were 0.4 mm or more in long axis, and 0.3     mm or more in short axis. The letter n represents the number of ovaries. 

                  TABLE 2                                                         ______________________________________                                        Germinal Vesicle Breakdown in Rat Graafian                                    Follicle Ova after PMSG and IL-8 Administration                               Time after IL-8                                                                            Number of Germinal Vesicle Breakdown                              Administration (hour)                                                                     Observed Ova                                                                            Number    Percentage                                   ______________________________________                                         4 [n = 1]  38          2         5.3                                         10  [n = 3]          106                                                                                       57                                                                                        53.8                             20  [n = 3]          104                                                                                       24                                                                                        23                               ______________________________________                                         The examined graafian follicles were 0.4 mm or more in long axis, and 0.3     mm or more in short axis. The letter n represents the number of ovaries.      IL8: Interleukin8                                                        

As is obvious from the results, interleukin-8 has an activity of causingequivalent ovum maturation to hCG; and promoting resumption of meiosisin a mature follicle, germinal vesicle breakdown, first polar bodyrelease through the metaphase, anaphase and telophase of the firstmeiosis, and the progress from the metaphase of the second meiosis tothe interphase of the second meiosis.

Example 6 Effects of Interleukin-8 in vitro on Cumulus-oocyte ComplexExpansion

(1) Method

Rats were dosed with PMSG, and 48 hours later, the ovaries wereisolated. The cumulus-oocyte complexes were then individually harvestedfrom Graafian follicles by puncture, dispersed in Eagle's MinimumEssential Medium (MEM, Nissui Pharmaceutical Co., Ltd., Japan),recovered using a micropipette, and transferred into 50 μl of MEM, thesurface of which was covered with a silicon oil (Aldrich Chemicals Co.,USA) to prevent evaporation. The resultant was then incubated in amedium without addition of serum at 37° C. for 20 hours in an atmosphereof 5% carbon dioxide gas and 95% air. Interleukin-8 was added in anamount of 100 ng/ml.

(2) Results

In the presence of serum, a cumulus-oocyte complex will exhibit cumulusexpansion through in vitro incubation for approximately 20 hours. On theother hand, it is known that the cumulus expansion reaction does notoccur in the absence of serum, and this fact was reproduced in thisexperiment. Nevertheless, cumulus expansion or mucus secretion wasinduced by incubation with addition of interleukin-8 even in the absenceof serum. Accordingly, interleukin-8 has been revealed to directly acton a cumulus-oocyte complex and exhibit effects as described above.

Example 7 Distribution of Interleukin-8 in the Ovarian Tissue andOvarian Cells

Immunostaining at tissue and cell level was performed in order toclarify distribution and production sites of interleukin-8 in the ovary.

(1) Method

From Wister rats (clean grade), ovaries were isolated and made intoserial sections of 4 μm in thickness according to the process in Example5. These sections were then incubated together with polyclonal rabbitantibodies raised against rat interleukin-8 (10 mg/ml) at 4° C.overnight. Subsequently, the sections were washed with phosphatebuffered saline (pH 7.2, hereinafter referred to as PBS) 5 times, andincubated together with a 100-fold-diluted fluoresceinisothiocyanate-rabbit immunoglobulin G conjugate solution at 23° C. for3 hours in a dark place. The sections were then washed with PBS again,and mounted with a glycerin-PBS (1:1, v/v) solution to be subjected toobservation with a fluorescence microscope (Axiophoto, Carl Zeiss,Germany).

Preparation of granulosa cells and thecal interstitial cells was carriedout according to the method of Li, Y-X, et al. (Li, Y-X, et al., Mol.Cell. Endocrinol., 54, 221, 1987). The procedure will be brieflydescribed below. A subject ovarian follicle was punctured using a 28gauge subcutaneous injection needle, and the contents were removed anddispersed into MEM. Granulosa cells without cumulus-oocyte werecollected by centrifugation. The remaining ovary was sufficientlypunctured using the same needle, and washed several times to removeresidual granulosa cells. Afterward, the ovarian tissue was incubated ina 0.08% collagenase solution at 37° C. for 5 min. The dissociated cellswere discarded, and the ovarian tissue fragments were further incubatedin a 0.4% collagenase solution at 37° C. for 1 hour to collect thecalinterstitial cells. The obtained cells were dispersed in MEM containing5% fatal bovine serum at a density of 1×10⁵ cells/ml, seeded in a 4-wellLabtech chamber (Nunk, Naperville, Ill.), and incubated in a 5% carbondioxide gas incubator. Twenty four hours after the start of incubation,the medium was changed to the fresh medium without serum. Afterincubating for further 48 hours, the resultant was subjected toimmunostaining according to the procedure described above.

(2) Results

Interleukin-8 was found to be distributed in theca cells of ovariesbefore and after ovulation. The intensity of staining was increased byhCG administration. The distribution of interleukin-8 in a ovarianfollicle was not clear. In response to the growth of the ovarianfollicle, the growing follicle was locally stained at the interleukin-8distribution sites in the theca cell layers. Theca cells in an immatureovary of a rat were incubated in the absence of serum in order toexamine whether or not interleukin-8 is produced in an immature ovary.As a result, interleukin-8 was found to be present in the supernatant byELISA and SDS-PAGE analysis. Interleukin-8 was revealed to be locallypresent especially and mostly in the cytoplasm by histologicalobservation.

The above-described results obviously show that ovarian endogenousinterleukin-8 plays an important role on normal ovum growth, and thatthe theca cells can reliably be considered as cells producing endogenousinterleukin-8.

Industrial Applicability

According to the present invention, leukocyte chemotactic factors havebeen revealed to have proconceptive activities. In addition to promotingactivities for ovum maturation and ovarian follicle growth, such factorsalso exhibit activities of promoting implantation of the fertilized ovumonto the endometrium. Accordingly, it has been shown that such factorscan be used in drugs or treatment agents in relation to medicaltreatment for infertility and artificial insemination in human beingsand animals.

We claim:
 1. A proconceptive method comprising administeringinterleukin-8 or monocyte chemotactic and activating factor in an amounteffective to increase implantation of fertilized ovum or to promotematuration of unfertilized ovum.